The invention relates to vaccine vectors comprising bacteria.
The use of vaccines is a cost-effective medical tool for the management of infectious diseases, including infectious diseases caused by bacteria, viruses, parasites, and fungi. In addition to effecting protection against infectious diseases, vaccines may now also be developed which stimulate the host""s immune system to intervene in tumor growth.
Host immune responses include both the humoral immune response involving antibody production and the cell-mediated immune response. Protective immunization via vaccine has usually been designed to induce the formation of humoral antibodies directed against infectious agents, tumor cells, or the action of toxins. However, the control of certain diseases characterized by the presence of tumor cells or by chronic infection of cells with infectious agents, often requires a cell-mediated immune response either in place of, or in addition to the generation of antibody. While the humoral immune response may be induced using live infectious agents and agents which have been inactivated, a cellular immune response is most effectively induced through the use of live agents as vaccines. Such live agents include live infectious agents which may gain access to the cytoplasm of host cells where the proteins encoded by these agents are processed into epitopes which when presented to the cellular immune system, induce a protective response.
Microorganisms, particularly Salmonella and Shigella which have been attenuated using a variety of mechanisms, have been examined for their ability to encode and express heterologous antigens (Coynault et al., 1996, Mol. Microbiol. 22:149-160; Noriega et al., 1996, Infect. Immun. 64:3055-3061; Brett et al., 1993, J. Immunol. 150:2869-2884; Fouts et al., 1995, Vaccine 13:1697-1705, Sizemore et al., 1995, Science 270:299-302). Such bacteria may be useful as live attenuated bacterial vaccines which serve to induce a cellular immune response directed against a desired heterologous antigen.
Listeria monocytogenes (L. monocytogenes) is the prototypic intracellular bacterial pathogen which elicits a predominantly cellular immune response when inoculated into an animal (Kaufmann, 1993, Ann. Rev. Immunol. 11:129-163). When used as a vector for the transmission of genes encoding heterologous antigens derived from infectious agents or derived from tumor cells, recombinant Listeria encoding and expressing an appropriate heterologous antigen have been shown to successfully protect mice against challenge by lymphocytic choriomeningitis virus (Shen et al., 1995, Proc. Natl. Acad. Sci. USA 92:3987-3991; Goossens et al., 1995, Int. Immunol. 7:797-802) and influenza virus (Ikonomidis et al., 1997, Vaccine 15:433-440). Further, heterologous antigen expressing recombinant Listeria have been used to protect mice against lethal tumor cell challenge (Pan et al., 1995, Nat. Med. 1:471-477; Paterson and Ikonomidis, 1996, Curr. Opin. Immunol. 8:664-669). In addition, it is known that a strong cell-mediated immune response directed against HIV-1 gag protein may be induced in mice infected with a recombinant L. monocytogenes comprising HIV-1 gag (Frankel et al., 1995, J. Immunol. 155:4775-4782).
Although the potential broad use of Listeria as a vaccine vector for the prevention and treatment of infectious disease and cancer has significant advantages over other vaccines, the issue of safety during use of Listeria is not trivial. The use of the most common strain of Listeria, L. monocytogenes, is accompanied by potentially severe side effects, including the development of listeriosis in the inoculated animal. This disease, which is normally food-borne, is characterized by meningitis, septicemia, abortion and often a high rate of mortality in infected individuals. While natural infections by L. monocytogenes are fairly rare and may be readily controlled by a number of antibiotics, the organism may nevertheless be a serious threat in immunocompromised or pregnant patients. One large group individuals that might benefit from the use of L. monocytogenes as a vaccine vector are individuals who are infected with HIV. However, because these individuals are severely immunocompromised as a result of their infection, the use of L. monocytogenes as a vaccine vector is undesirable unless the bacteria are fully and irreversibly attenuated.
There is a need for the development of a strain of L. monocytogenes for use as a vaccine in and of itself and for use as a bacterial vaccine vector which is attenuated to the extent that it is unable to cause disease in an individual into whom it is inoculated. The present invention satisfies this need.
The invention includes a method of eliciting a T cell immune response to an antigen in a mammal comprising administering to the mammal an auxotrophic attenuated strain of Listeria which expresses the antigen, wherein the auxotrophic attenuated strain comprises a mutation in at least one gene whose protein product is essential for growth of the Listeria. In a preferred embodiment, the Listeria is L. monocytogenes. In another preferred embodiment, the auxotrophic attenuated strain is auxotrophic for the synthesis of D-alanine. In addition, the mutation comprises a mutation in both the dal and the dat genes of the Listeria.
In one aspect of the invention, the auxotrophic attenuated strain further comprises DNA encoding a heterologous antigen, or the the auxotrophic attenuated strain further comprises a vector comprising a DNA encoding a heterologous antigen.
The heterologous antigen may be an HIV-1 antigen.
The invention also includes a vaccine comprising an auxotrophic attenuated strain of Listeria which expresses an antigen, wherein the auxotrophic attenuated strain comprises a mutation in at least one gene whose protein product is essential for growth of the Listeria.
In preferred-embodiments, the Listeria is L. monocytogenes. In other preferred embodiments, the auxotrophic attenuated strain is auxotrophic for the synthesis of D-alanine. In yet other preferred embodiments, the mutation comprises a mutation in both the dal and the dat genes of the Listeria.
The auxotrophic attenuated strain may further comprise DNA encoding a heterologous antigen or a vector comprising a DNA encoding a heterologous antigen.
The heterologous antigen may be an HIV-1 antigen.
Also included in the invention is an isolated nucleic acid sequence comprising a portion of a Listeria dal gene and an isolated nucleic acid sequence comprising a portion of a Listeria dat gene.
In addition, the invention includes an isolated strain of Listeria comprising a mutation in a dal gene and a mutation in a dat gene which render the strain auxotrophic for D-alanine. In one aspect, the isolated strain of Listeria further comprises a heterologous antigen.